Direct-Fed Microbial

ABSTRACT

An isolated microorganism comprising a Propionibacteria strain is described. When the microorganism is fed to a ruminant, protein and fat levels in milk produced by the ruminant are increased, while body condition and milk production levels are maintained. When fed to the ruminant, the microorganism also has positive effects on various metabolic hormones and metabolites, e.g, an increase in energy balance, plasma non-esterified fatty acids levels, and plasma leptin level. Supplementation with propionibacteria reduced dry matter intake but did not affect milk production in the cows. Therefore, the propionibacteria of the invention made the cows more energy efficient as cows produced the same amount of milk, yet consumed less dry matter.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/980,878, which is adivisional of U.S. patent application Ser. No. 09/912,049, filed Jul.24, 2001, now U.S. Pat. No. 6,951,643, the entireties of both of whichare incorporated herein by reference.

BIBLIOGRAPHY

Complete bibliographic citations of the references referred to herein bythe first author's last name in parentheses can be found in theBibliography section, immediately preceding the claims.

FIELD OF THE INVENTION

The invention relates to microorganisms for ingesting by animals. Moreparticularly, though not exclusively, the present invention relates tomicroorganisms that are useful as a direct-fed for ruminants.

DESCRIPTION OF THE RELATED ART

Milk solid components include protein, fat, lactose, and minerals. Milkprotein has economic value because, for example, higher protein leads tohigher cheese yields. Furthermore, in recent years, consumers havebecome increasingly concerned about the effects of dietary fatconsumption on their health. Low fat milk and low fat cheese have becomepopular. In many countries, including the United States, the payment formilk shipped to cheese plants has changed to a system based on bothprotein and fat content from one based on milk fat. This market trendincreases the emphasis on milk protein. However, milk fat continues tobe an important component in some markets were it is used to make icecream and butter. In these markets, a premium of $2 per pound is paidfor milk fat.

Milk protein represents about 3% to about 4% of the total content ofmilk, depending on numerous factors, including animal breed and diet.Milk protein is synthesized in the mammary gland from amino acids. Thebiological control mechanism of milk protein synthesis is still largelyunknown. Milk protein requires a supply of the appropriate amino acidsas well as a source of energy at the mammary gland.

Ruminal volatile fatty acids affect the concentration of fat and proteinin milk. In general, increasing propionate production increases theconcentration of protein in milk, while increasing acetate increases theconcentration of fat in milk.

The volatile fatty acids are the major precursors of glucose, which isused to create energy for the physiological processes in the animal.Dairy cattle fed typical diets high in starch produce volatile fattyacids in the following proportions: 58% acetate, 30% propionate, and 12%butyrate. Propionate production conserves 109% of the energy fromglucose, while acetate production conserves only 77%.

Energy balance is the difference between the amount of energy consumedby an animal and the amount of energy expended by the animal. The energybalance of an animal can be in a positive or negative state, and it canbe measured. The effect of dietary protein and energy supply on milkprotein synthesis is affected by rumen fermentation. Dietary proteinsare broken down to their constituent amino acids during digestion. Theamino acids are absorbed into the body. Carbohydrates in the diet aredegraded by the rumen microorganisms to volatile fatty acids, which arethe major energy supply for the cow.

Many high producing dairy cows are unable to consume enough feed to meetenergy demands during early postpartum lactation, resulting in a stateof negative energy balance. Energy balance (EB) is quantified usingmeasures of dry matter intake (DMI), milk production (quantity andcomposition), and body weight (BW) and may be associated withreproductive efficiency. In lactating dairy cows, EB during the firstfew weeks postpartum is positively related to concentrations of plasmaprogesterone (P₄) during the first postpartum estrous cycle (Berghorn etal., 1988; Villa-Godoy et al., 1988; Spicer et al., 1990). Cowsexhibiting estrus with subsequent formation of a functional corpusluteum that secretes maximal P₄ levels have the best chance ofmaintaining pregnancy (Villa-Godoy et al., 1988). In addition, cows thatexpress estrus before first postpartum ovulation have greater EB thancows that do not express estrus (Berghorn et al., 1988; Spicer et al.,1990). Negative EB is therefore a likely cause for poor reproductiveefficiency in lactating dairy cows (Kimura et al, 1987; Sklan et al.,1991).

Although studies implicate EB as a regulator of ovarian function, thehormones or metabolites mediating the effect of EB are unclear. Plasmacholesterol (Carroll et al., 1990) and insulin (Koprowski and Tucker;1973; Smith et al., 1978) increase whereas plasma non-esterified fattyacids (NEFA) decrease (Staples and Thatcher, 1990; Canfield and Butler,1991; Beam and Butler, 1998) with increasing week of lactation.Concentrations of cholesterol in blood of cattle are modified byvariations in fat, protein and (or) energy intake and increase as EBincreases (Kronfeld et al., 1980; Grummer and Carroll 1988; Ronge etal., 1988; Spicer et al., 1990; 1993). Because insulin in vitrostimulates mitogenesis and steroidogenesis of bovine ovarian cells(Schams et al., 1988; McArdle et al., 1989; McArdle et al., 1991;Saumande et al., 1991; Spicer, et al, 1993, Gong et al., 1994; Spicerand Chamberlain, 1998), negative EB may affect ovarian activity bydecreasing luteal progesterone (P₄) production (Talayera et al., 1985;Grummer and Carroll, 1991; Spicer et al., 1993; Hawkins et al., 1995).

Propionate, a ruminal volatile fatty acid, acts as a precursor forhepatic glucose production. Glucose is used to create energy for thephysiological processes in the animal. Drenching the diet of lactatingcows with calcium propionate elevates plasma glucose concentration(Jonsson et al., 1998). Conversely, preventing reabsorption of glucosein renal tubules decreases plasma glucose and insulin in dairy cows(Amaral-Phillips et al., 1993). Also, infusion of butyrate, a ruminalvolatile fatty acid that inhibits the use of propionate forgluconeogenesis into the rumen of lactating cows, decreases plasmaglucose concentrations (Huhtanen et al., 1993). Whether plasma insulin,IGF-I, cholesterol, and other metabolites are altered by changes inruminal propionate is unknown.

Propionibacteria are natural inhabitants of the rumen that comprise 1.4%of the ruminal microflora and produce propionic and acetic acid in therumen (Oshio et al., 1987). Directly feeding propionibacteria mayincrease hepatic glucose production via increased in ruminal propionateproduction and absorption. The efficiency of utilization for maintenanceof metabolizable energy of propionic acid is 0.86 vs. 0.59 for acetateand 0.76 for butyrate (McDonald et al., 1987). Organisms of the genusPropionibacterium comprise a small proportion of the ruminal microfloraand are slow growing. Propionibacteria are an industrially importantgroup of organisms primarily used by the dairy-food industry as startercultures for Swiss-type cheeses. Other industrial applications of thepropionibacteria have been described including their use in theproduction of vitamin B12 and propionic acid and as inoculants forsilage and grain. Other applications of the propionibacteria includetheir use as direct-fed microbials. However, little research has beenreported to date.

With the adoption of recent economic incentives for producing milk of adesired composition, dairy farmers can realize an economic benefit byfeeding specific dietary enhancements to manipulate ruminalfermentation. Therefore, dairy farmers will benefit from products thatcan successfully control or manage ruminal microbial fermentationactivity.

In view of the foregoing, it would be desirable to provide a direct-fedmicrobial which, when fed to ruminants, increases the protein and fatlevels in milk produced by the ruminant while maintaining body conditionand milk production levels. It would also be desirable for themicroorganism, when fed to livestock, to have a positive effect onvarious metabolic hormones and metabolites.

SUMMARY OF THE INVENTION

The invention, which is defined by the claims set out at the end of thisdisclosure, is intended to solve at least some of the problems notedabove. Isolated Propionibacteria strains are provided. In a preferredembodiment, the strains are P. acidipropionici or P. jensenii.

A method of feeding a ruminant the microorganism is also provided.Feeding ruminants the microorganism increases at least one of energybalance, plasma non-esterified fatty acids levels, and plasma leptinlevel in the ruminant fed the microorganism when compared to therespective energy balance, plasma non-esterified fatty acids levels, andplasma leptin level in the ruminant when not fed the microorganism. Milkfrom ruminants fed the microorganism has a higher percent of proteinthan the percent of protein in milk produced by the ruminant when notfed the microorganism. The milk also has a higher fat level whencompared to milk produced by the ruminant when not fed themicroorganism.

A feed composition is also provided. The feed composition includes theisolated microorganism described above and a carrier.

A method of forming a direct fed is also provided. The method includessteps of growing, in a liquid nutrient broth, a culture including theisolated microorganism described above, and separating the microorganismfrom the liquid nutrient broth. The separated microorganism can then befreeze-dried and added to a carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred exemplary embodiments of the invention are illustrated in theaccompanying drawings.

FIGS. 1 and 2 show pulsed-field gel electrophoresis analysis of Xba Idigested genomic DNA of Propionibacterium strains. Strainidentifications are shown.

FIG. 3 shows weekly changes in percent milk protein of postpartum cowsfed Propionibacteria (n=9) and control (n=10) diets during the first 12wk of lactation. Means with different superscript within week differ(P<0.01). SEM=0.14 for control and 0.15 for treatment cows.

FIG. 4 illustrates weekly changes of percent solids-non-fat ofpostpartum cows fed Propionibacteria (n=9) and control (n=10) dietsduring the first 12 wk of lactation. Means with different superscriptwithin week differ (P<0.05). SEM=0.14 for control and 0.15 for treatmentcows.

FIG. 5 displays weekly changes in percent milk fat of postpartum cows.Data from cows fed (see attached for new figure.) Propionibacteria (n=9)and control (n=10) diets during the first 12 wk postpartum. SEM was 0.08for control and 0.08 for treatment cows. *Means within week differ(P<0.13).

FIG. 6 displays weekly changes in dry matter intake (DMI) expressed as gdry matter per kg of body weight (BW) of postpartum cowsPropionibacteria (n=9) and control (n=10) diets during the first 12 wkpostpartum. SEM was 0.8 for control and 0.8 for treatment cows. *Meanswithin week differ (P<0.01).

FIG. 7 shows weekly changes in fat correct milk of postpartum cows fedPropionibacteria (n=9) and control (n=10) diets during the first 12 wkpostpartum. There was no significant effect of treatment over the 12 wk(P>0.7).

FIG. 8 shows changes in plasma non-esterified fatty acids (NEFA)concentrations of postpartum cows during the first 12 wk of lactation.Data from cows fed Propionibacteria (n=9) and control (n=10) diets.Means without a common superscript differ (P<0.01). SEM=47 for controland 49 for treatment cows.

FIG. 9 illustrates weekly changes in plasma leptin concentrations ofpostpartum cows fed Propionibacteria (n=9) and control (n=10) dietsduring the first 12 wk of lactation. *Mean within week differs (P<0.10)from Propionibacteria mean. Pooled SEM=1.1 for control and 1.1 forPropionibacteria treated cows.

FIG. 10 demonstrates weekly changes in energy balance of postpartum cowsfed Propionibacteria (n=9) and control (n=10) diets during the first 12wk postpartum. SEM was 1.48 for control and 1.54 for treatment cows.*Means within week differ (P<0.10).

Before explaining embodiments of the invention in detail, it is to beunderstood that the invention is not limited in its application to thedetails of construction and the arrangement of the components set forthin the following description or illustrated in the drawings. Theinvention is capable of other embodiments or being practiced or carriedout in various ways. Also, it is to be understood that the phraseologyand terminology employed herein is for the purpose of description andshould not be regarded as limiting.

DETAILED DESCRIPTION

In accordance with the present invention, there may be employedconventional molecular biology and microbiology within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,Third Edition (2001) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

Microorganisms:

Propionibacteria strains useful in the invention were selected based onseveral criteria. One or more of the following criterion were used toselect useful propionibacteria strains: 1) production of at least 0.9%propionate (vol/vol) in sodium lactate broth (NLB) and at least 0.2%propionate (vol/vol) in rumen fluid (in vitro), 2) isolation from therumen, 3) ability to survive and grow in the rumen, 4) ability to begrown commercially, and 5) ability to survive a freeze-drying process.All of the strains possessing one or more of these properties had agenetic profile of group 1 (as defined below). Strains possessing one ormore of these properties were then tested in vivo. When fed toruminants, the tested strain increased protein and fat levels in milkproduced by the ruminants, while maintaining body condition and milkproduction levels. When fed to ruminants, the tested strain increasedplasma leptin levels and decreased dry matter intake, making thelactating cows more energetically efficient.

Preferably, the microorganisms of the invention are selected from thegenus Propionibacterium. The microorganisms were isolated from the rumenof fistulated ruminants. Multiple collections of rumen fluid wereobtained over a period of time. Colonies were isolated. The coloniesthat were suspected of being Propionibacteria were then grown in broth,and the strain of the isolates was determined based on biochemical testsand carbohydrate fermentation patterns. Based on carbohydratefermentation patterns and biochemical tests, 95% of the isolates wereidentified as P. acidipropionici and the remaining 5% were identified asP. jensenii.

The plasmid contained within each isolate was examined to determinewhether plasmids carried by the isolates had an effect on survival andfunction in ruminant environments. Overall, 35% of the 132 isolatesexamined contained plasmids. Two plasmid profiles were common, a single2.5 kb plasmid and a single 7.0 kb plasmid. Only one strain was found tocontain more than one plasmid. The predominant plasmid profile varied atdifferent sampling times.

The intact genomic DNA from the isolates was also examined to determinethe genetic diversity of the strains and to reduce the number of strainssubsequently tested in feeding experiments. Pulsed-field gelelectrophoresis analysis of genomic DNA identified 13 distinct Xba Ifragment patterns. However, only one strain was predominant in the rumenof all cows throughout the sampling times.

The isolates were then tested for volatile fatty acid production.Various fatty acids such as propionate, acetate, butyrate, and lactatewere detected and concentration determined. An isolate that produced thehighest amount of propionate under conditions similar to the rumen wasselected for animal testing.

In a preferred embodiment, the microorganism is of the genusPropionibacterium and more preferably P. acidipropionici and P.jensenii. Preferred strains of bacteria include P. acidipropionici andjensenii strains P169, P170, P179, P195, and P261, especially strain,P169. The P169 and P170 strains are available from the microorganismcollection of the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110, under accession numbers ATCCPTA-5271 and ATCC PTA-5272, respectively, and were deposited on Jun. 18,2003. Strains P179, P195, and P261 were deposited on Apr. 2, 2008 at theAgricultural Research Service Culture Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill., 61604 and given accession numbers NRRLB-50133, NRRL B-50132, and NRRL B-50131, respectively. All of thepreferred strains were found to have group 1 genomic profiles (asdefined below). Therefore, other strains of P. acidipropionici or P.jensenii that have a group 1 genomic profile and which have a commonidentifying characteristic of successful performance in the presentinvention are also preferred strains. These other strains are referredto hereinafter as “genetic equivalents.”

Direct Feed Assays on Cows:

Strain P169 was used in animal trials to determine the effects of thedirect-fed propionibacteria on energy balance, milk yield andcomposition, metabolites and hormones of early lactating dairy cows.

In a first study in which cows were fed freeze-dried propionibacteriaculture, in the control group, there was a decline in rumen pH duringthe 11.5 h after the morning allocation of concentrate (−0.486 pHunits). In contrast, in the cows that received the Propionibacterium,the decline in rumen pH occurred during 6 h after the morning feed(−0.32 pH units) and then remained relatively stable before returning toa prefeeding value. Thus, the Propionibacterium had a physiologicaleffect on the cows.

In a second animal study, nineteen pluriparous Holstein cows wereindividually fed a total mixed ration from −2 to 12 wk postpartum. Eachtreated cow received 17 g of a 1:10 preparation of the freeze-driedpropionibacteria culture, which was at a concentration of about 3.5×10¹⁰CFU/g, and maltodextrin carrier daily. Daily feed intake and milkproduction and weekly body weight were recorded. Blood samples werecollected twice a week for quantifications of plasma cholesterol,non-esterified fatty acids (NEFA), leptin, insulin, and glucose. Whencompared to control cows, cows supplemented with Propionibacteriaculture improved energy balance (EB) and body weight at the first weekof lactation. Supplementation with propionibacteria reduced dry matterintake (DMI) when expressed as g DMI per kg body weight, but did notaffect milk production in the cows. Therefore, the propionibacteria madethe cows more energy efficient as cows produced the same amount of milk,yet consumed less dry matter. When compared to control cows, cows fedPropionibacteria had greater percentages of milk protein andsolids-non-fat (SNF) during the first week of lactation and in addition,the percentages of milk fat increased over the 12 weeks of observation.Thus, due to economic incentives for milk fat, feeding cowspropionibacteria of the invention provides economic advantages to dairyfarmers. Plasma glucose, insulin, and cholesterol concentrations werenot significantly affected by supplemental feeding of Propionibacteriaculture. Thus, there was no negative effect on these parameters.

Plasma NEFA concentrations at week 1 of lactation was significantlylower in control than treated cows but not thereafter. Decreasing plasmaNEFA concentrations with week postpartum may be an indication that thecows are moving towards positive EB.

Manipulation of the ruminal fermentation to increase propionate improvesthe energetic efficiency of the animal. As energy efficiency isincreased, a positive energy balance is obtained, which has a directeffect on reproductive efficiency. Cows with a positive energy balancehave a greater chance of maintaining pregnancy, which is necessary toeffectively increase milk production over the life of the cow.

Leptin, which is a neurotransmitter produced by fat cells and involvedin the regulation of appetite, was significantly higher in treated cowsthan control cows throughout the study. Leptin from adipocytes passesfrom circulation to the cerebrospinal fluid and to the hypothalamus andmay affect satiety. Thus, the direct fed had positive effect on cows'plasma leptin levels, which may be an important signal for regulation offeed consumption that indirectly influences milk production, energystatus, and reproductive functions.

In sum, feeding Propionibacteria culture strain P169 to early lactatingdairy cows improved some production parameters of lactation but withoutnegatively impacting reproductive function.

Preparation and Feeding of Direct-Fed Propionibacteria:

In a preferred embodiment, the microorganism is fed to a ruminant, andthe microorganism becomes established in the rumen. Preferably, theamount of the microorganism that is delivered to the ruminant is about6×10⁹ CFU to about 6×10¹² CFU/animal/day. This translates intoapproximately 1×10⁵ to 1×10⁸ CFU/ml of rumen fluid for an averaged sizedcow. In a more preferred embodiment, about 6×10¹¹ CFU/animal/day of themicroorganism is delivered to the ruminant. In a preferred embodiment,the microorganism is fed to a ruminant such that the microorganismbecomes established in the rumen at a level of about 1×10⁵ CFU per ml ofruminen fluid to about 1×10⁸ CFU per ml of ruminen fluid.

The microorganism of the present invention may be presented in variousphysical forms, for example as a top dress, liquid drench, gelatincapsule, or gels. In a preferred embodiment of the top dress form of themicroorganism, freeze-dried Propionibacterium fermentation product isadded to a carrier, such as whey, maltodextrin, sucrose, dextrose,limestone (calcium carbonate), rice hulls, yeast culture, dried starch,sodium silico aluminate. In a preferred embodiment of the liquid drench,freeze-dried Propionibacterium fermentation product is added to acarrier, such as whey, maltodextrin, sucrose, dextrose, dried starch,sodium silico aluminate, and a liquid is added to form the drench. In apreferred embodiment of the gelatin capsule form, freeze-driedPropionibacterium fermentation product is added to a carrier, such aswhey, maltodextrin, sugar, limestone (calcium carbonate), rice hulls,yeast culture dried starch, sodium silico aluminate. ThePropionibacteria and carrier are enclosed in a rumen degradable gelatincapsule. In a preferred embodiment of the gels form, freeze-driedPropionibacterium fermentation product is added to a carrier, such asvegetable oil, sucrose, silicon dioxide, polysorbate 80, propyleneglycol, butylated hydroxyanisole, citric acid, and artificial coloringto form the gel.

In one preferred embodiment of the present invention, a microorganism isdirectly fed to a ruminant to increase the protein and fat concentrationin milk produced by animals fed the microorganisms and to have apositive effect on various metabolic hormones and metabolites.

In a preferred embodiment, the P. acidipropionici is fermented to a5×10⁸ CFU/ml to a 4×10⁹ CFU/mllevel with a level of 2×10⁹ CFU/ml beingmore preferred. The bacteria are harvested by centrifugation, and thesupernatant is removed. The pelleted microorganisms can then be fed tothe ruminant. Preferably, the pelleted microorganisms are freeze-driedfor direct feeding to the ruminant.

In a preferred embodiment, the microorganisms are added to animal feed.Preferably, the microorganism is fed as mixture of freeze-driedmicroorganism, which is at a concentration of about 3.5×10¹⁰ CFU/g and acarrier, which preferably is a maltodextrin carrier. Preferably, about17 g of the 1:10 mixture is fed to each animal each day. In a preferredembodiment, the microorganism is fed from 2 weeks prior to parturitionto 12 weeks postpartum, although the microorganism can be fed fordifferent durations and at different times.

EXAMPLES

The following Examples are provided for illustrative purposes only. TheExamples are included herein solely to aid in a more completeunderstanding of the presently described invention. The Examples do notlimit the scope of the invention described or claimed herein in anyfashion.

Example 1 Isolation of Propionibacteria

The propionibacteria strains examined in this study were obtained fromrumen fluid collected from five fistulated Holstein cows at the OklahomaState University Dairy Cattle Research Center. Rumen fluid was collectedthree times over a five month period, spanning 150 days of the 305 daylactation. The cows received a ration concurrent with the pounds of milkproduced daily (See Table 1).

TABLE 1 Ration Content Ration Mix % as fed Pen A % as fed Pen B Alfalfa7.8 2.2 Sorghum 44.6 66.4 Whole corn 7.5 2.1 Grain mix 38.0 27.1 Shelledcorn (67%) Soybean meal (27%) Molasses (3%) Limestone (1%) Dicalciumphosphate (1%) Trace minerals (1%) Prairie hay 2.1 2.2 Bypass protein 1lb ** Cow 179 fed Pen A Ration, all others Pen B ration.

At each sampling time approximately 100-150 ml rumen fluid was collectedfrom beneath the mat of ingesta or squeezed from the ingesta into 250 mlbottles. The fluid was transported to the laboratory and strainedthrough cheesecloth to remove the feedstuff debris. The fluid was thendiluted and plated onto a selective agar containing erythritol as thecarbon source with the pH indicator bromocresol purple with selectiveantibiotics. All plates were incubated for 7-10 days at 32° C. underanaerobic conditions (CO₂ GasPak®, B-D Laboratories, Inc. EastRutherford, N.J.). After 7-10 days, the plates were examined for smooth,raised, convex colonies, which fermented erythritol as indicated by a pHchange in the agar medium. Propionibacteria colonies were yellow due tothe bromocresol purple indicator changing from purple to yellow as theerythritol was fermented and acid was formed. Individual colonies werepicked from the original serial dilution plates of rumen fluid andstreaked on four consecutive streak plates to ensure purity.

Isolated colonies were picked into 10 ml tubes of sodium lactate broth(NLB) and incubated statically for 36-48 hours at 32° C. The cultureswere routinely propagated from 1% transfers in NLB. Cultures can bestored as frozen stocks at −75° C. in, for example, NLB with 10.0%glycerol.

Identification of Propionibacteria Isolates:

Colonies suspected of being propionibacteria colonies were grown in 10ml NLB tubes for 36 hours at 32° C. After incubation, the isolates weregram stained and tested for catalase production. Gram positive,pleomorphic rods with characteristic V or Y configurations were testedfor their ability to ferment lactose, mannitol, and trehalose. Furtheridentification was made by observing the reduction of esculin (0.01%)and nitrate (0.2%) as well as the hydrolysis of gelatin (12%). Grampositive isolates that reduced esculin and nitrate were classified as P.acidipropionici and non-nitrate reducing Gram positive, esculin reducerswere classified as P. jensenii. It should be noted that the biochemicaltests used to distinguish P. acidipropionici from P. jensenii is not100% conclusive. For example, occasionally, when tests are repeated, thespecies classification changes. The genomic DNA grouping described belowis a more accurate classification method.

The isolates were tested for volatile fatty acid production using aHewlett-Packard® 1090 HPLC. The isolates were grown from a 1% transferin 10 ml NLB incubated for 36 hours at 32° C. The cells were removedfrom the media by centrifugation (1500×g 15 min) and the supernatant wasthen filtered through 0.2 um Gelman® filter and mixed with equal volumesof 0.01 M H₂SO₄. 1 ml samples were injected (5 ul) and separated using aHPX-87H column (BioRad®) preheated to 65° C. with 0.005 M H₂SO₄ as themobile phase at a flow rate of 1 ml/min. Propionate, acetate, butyrate,and lactate were detected using a diode array detector scanningwavelengths 210-254 nm. Concentrations were determined by calculatingpeak areas and comparing these to known areas of external standardsusing Hewlett-Packard® software.

A total of 132 strains isolated from the rumen fluid were identified asPropionibacterium based on a gram-positive, pleomorphic cell morphology,the reduction of esculin, hydrolysis of gelatin, and production ofpropionate and acetate from lactate. All isolates examined in this studyfermented mannitol and trehalose. All but three of the isolatesfermented lactose. A total of 126 strains reduced nitrate and 26 ofthese isolates were found to reduce nitrite as well. Based on theseresults, 126 of the isolates were identified as P. acidipropionici(capable of nitrate reduction), and the remaining five were identifiedas P. jensenii. It should be noted that the tests used to distinguish P.acidipropionici from P. jensenii is not one hundred percent conclusive.Occasionally, when tests are repeated, the species classificationchanges. The genomic DNA grouping described below is a more accurateclassification method.

Ruminal populations of propionibacteria ranged from 10³ to 10⁴ CFU/ml.Propionibacteria populations varied among different cows at differentsampling times. Three of the five cows used in this study had detectablepropionibacteria populations at two of the sampling times while one cowhad detectable propionibacteria populations at all three sampling times.Only one cow did not have detectable populations of propionibacteria atany of the sampling times. Isolates characterized as P. acidipropioniciwere predominant at all sampling times and accounted for 96% of thetotal propionibacteria isolated from the rumen.

Strains P169, P170, P179, P195, and P261 produced at least 0.9%(vol/vol) propionate in sodium lactate broth (NLB) and at least 0.2%(vol/vol) propionate in rumen fluid (in vitro).

Plasmid DNA Isolation:

In an effort to determine whether plasmids carried by the isolates hadan effect on survival and function in ruminant environments, plasmid DNAwas isolated from the propionibacteria strains and DNA analysis wasperformed. The DNA was resuspended in 40 ul Tris-EDTA buffer and 5 ultracking dye and then loaded onto an agarose gel. The DNA was separatedby gel electrophoresis using a 0.7% agarose gel at 50 volts. The agarosegels were then examined after 45 minutes of staining in ethidium bromidesolution.

Referring to Table 2 below, plasmids were detected in 32.6% (43 out of132) of the isolates screened. Only one strain contained more than oneplasmid. Of the 42 isolates that contained a single plasmid, 31 strainscontained a 2.7 kb plasmid and the remaining 11 strains contained a 7.0kb plasmid. There was no apparent relationship between plasmid contentand the biochemical and fermentation activity of the isolates. Inaddition, it is not known whether plasmids of the same molecular weightfound in different isolates are in fact identical.

TABLE 2 Plasmid DNA analysis of propionibacteria isolates. PlasmidContent Sampling Number of Number of Number of Date Isolates ScreenedIsolates Plasmids MW (kb) February 31 20 0 all 7.0 11 1 April 35 31 1all 2.7 4 0 June 66 65 0 1.6, 1.8 1 2

The predominant plasmid profile was different at each sampling time(Table 2). Plasmids of similar molecular weight were not detected atdifferent sampling times. The absence of plasmids in the majority ofstrains (67.4%) and the lack of conserved plasmids among isolates atdifferent sampling times may indicate the plasmids detected are notimportant for survival and function in the ruminal environment.

Preparation of Intact Genomic DNA:

In an effort to determine the genetic diversity of the strains and toreduce the number of strains subsequently tested in direct feedexperiments, intact genomic DNA from representative strains was isolatedfrom cells embedded in agarose beads. Cultures were grown to mid-logstage in 10 ml NLB with supplemented varying percentages of glycine. Thecells were harvested by centrifugation (9000×g for 10 min) andresuspended to one-tenth the original volume in 10×ET buffer (*500 mMEDTA, 10 mM Tris-HCl, pH 8.0). The cell suspension was mixed with anequal volume of 1% low-melting point agarose (Beckman Instruments, PaloAlto, Calif.), loaded into a syringe, and injected into Tygon® tubing(ID- 1/16″, OD-⅛″) where it was allowed to solidify. The solidifiedcell-agarose mixture was forced through the tubing into cold 10×ETbuffer and chopped into smaller pieces (beads). The beads were harvested(5500×g for 10 min), resuspended in 10 ml 10×ET containing 5 mg/mllysozyme and incubated at 32° C. with gentle shaking for 2 hours. Afterincubation, the beads were harvested by centrifugation (5500×g for 10min) and resuspended in 10 ml of lysis buffer (10×ET buffer containing100 ug/ml of Proteinase K and 1% Sarkosyl®), and incubated at 55° C. for15 hours to lyse cells and release the genomic DNA. After lysis, thebeads were harvested by centrifugation (5500×g for 10 min), resuspendedin 10 ml of 1 mM phenylmethylsulfonyl fluoride, and incubated at roomtemperature for 2 hours with gentle shaking to remove contaminatingprotease activity. The beads of purified DNA were washed three times inTE buffer (10 mM Tris-HCl, 1 mM EDTA-Na₂, pH 7.5), resuspended in 10 mlTE buffer, and stored at 4° C. until restriction endonuclease digestionwas performed.

In Situ Restriction Endonuclease Digestion and Pulsed-Field GelElectrophoresis of Genomic DNA:

Agarose beads containing DNA were equilibrated in 1× restrictionendonuclease buffer for 1 hour before enzyme digestion. Afterequilibration, 10-20 units of the restriction enzyme were added to 90 ulof beads and incubated at the appropriate temperature for 6-8 hours.Following digestion, the enzymes were inactivated by heating for 5minutes at 65° C. The melted beads were loaded onto a gel for fragmentseparation.

DNA fragments were separated on 1.0% agarose gels in 0.5×TBE buffer at15° C. for 23 hours using a CHEF-DRIII electrophoresis system (BioRad®).Each set of restriction endonuclease digests were separated at differentinitial and final pulse times to provide maximum separation of small,medium, and large fragments. To determine the molecular size of the DNAfragments lambda DNA multimers, intact yeast chromosomes and restrictionfragments of lambda DNA were included as standards.

Comparisons of genomic DNA profiles produced by Xba I digests identifiedisolates that shared the same DNA digestion pattern (FIGS. 1 and 2).Isolates with a common digestion pattern (>90% of the fragmentscomigrating) were assigned to the same genomic digestion profile group,which are shown below in Table 3. Overall, 21 different digestionprofiles were observed for the 132 isolates. Eight of the profiles wereunique to only one isolate. The predominant genomic profile (group 1)was shared by 48 isolates, which accounted for 43.6% of all isolatesexamined.

TABLE 3 Analysis of propionibacteria isolates. Genomic Plasmid Contentdigestion Isolate Species Number of profile Number IdentificationPlasmids MW (kb) group 162 P. acidipropionici 0 1 166 P. acidipropionici0 1 169 P. _acidipropionici 0 1 170 P. acidipropionici 0 1 173 P.acidipropionici 0 1 176 P. acidipropionici 0 1 178 P. acidipropionici 01 179 P. jensenii 0 1 180 P. acidipropionici 0 1 182 P. acidipropioniciND 1 188 P. acidipropionici 0 1 195 P. jensenii 1 7.0 2 233 P.acidipropionici 0 3 236 P. acidipropionici 1 2.7 4 238 P.acidipropionici 1 2.7 1 245 P. acidipropionici 1 2.7 1 246 P.acidipropionici 1 2.7 1 248 P. acidipropionici 1 2.7 1 249 P.acidipropionici 1 2.7 1 261 P. acidipropionici 1 2.7 1 266 P.acidipropionici 0 3 272 P. acidipropionici 1 2.7 1 277 P.acidipropionici 0 3 279 P. acidipropionici 1 2.7 1 345 P.acidipropionici 0 6 346 P. acidipropionici 0 10  347 P. acidipropionici0 10  348 P. acidipropionici 0 6 349 P. acidipropionici 0 6 350 P.acidipropionici 0 5 351 P. acidipropionici 0 6 352 P. acidipropionici 06 354 P. acidipropionici 0 5 362 P. acidipropionici 0 U 365 P.acidipropionici 0 U 377 P. acidipropionici 0 U 381 P. acidipropionici 0U 388 P. acidipropionici 0 U 393 P. acidipropionici 0 5 395 P.acidipropionici 0 U 400 P. acidipropionici 0 U ND = Not determined U =Unique genomic profile

Little diversity of the genomic digestion patterns was observed inisolates from the first two sampling periods. The group 1 digestionprofile was observed for 77.4% of the isolates obtained in the first twosampling periods. However, this digestion profile failed to be detectedin isolates from the June sampling. Other changes in the genomicprofiles of isolates from the June sampling were also detected. None ofthe genomic profiles observed in isolates from the first two samplingtimes were observed in the isolates obtained in the June sampling. Inaddition, the diversity of the genomic profiles increased from 2 and 3profiles detected in the first and second sampling periods, respectively(a total of 4 different profiles for the first two sampling periods) tomore than 9 different profiles detected in the June sampling with 8other isolates having unique profiles. The June sampling appears torepresent a major increase in the genetic diversity of the ruminalpropionibacteria that was not evident in either of the first twosampling times.

In Vitro Ruminal Models for Selection:

Rumen fluid was collected from two cannulated dairy cows 2 hours postfeeding. The rumen fluid was strained through 4 layers of cheese clothinto pre warmed (37° C.) thermos jugs. The rumen fluid was transportedback to the lab where it was again strained through 4 layers of cheesecloth. A 100 ml of strained rumen fluid was added to individual prewarmed (37° C.) flasks containing 100 mls of sterile Merten's buffer (1L dH₂O, 4.0 g ammonium bicarbonate, 35.0 g sodium bicarbonate). Strainedrumen fluid solids (10 g) were added to each flask.

Propionibacterium strains to be used in the in vitro fermentations weregrown in 10 ml tubes of sodium lactate broth (NLB) at 32° C. for 40 to48 hours. For inoculation of the flasks, 2.0 ml of a 40 to 48 hourPropionibacterium culture was added to duplicate flasks containing therumen fluid medium. The flasks were placed in a shaking water bath (37°C.). The mouth of each flask was sealed with a rubber stopper. Therubber stopper had 3 glass ports. Of the three ports, one port wasconnected (via rubber hosing) to a CO₂ tank to flush the flasks, oneport served as an exhaust to vent the CO₂ flush, and a third port wasused to remove samples for pH, VFA, and microbial analysis.

At hours 0, 6, 12, 24, 30, 36, 42, and 48 post inoculation, samples wereremoved from the flask through the sample ports for pH and volatilefatty acids analysis (VFA). The fluid collected for VFA analysis wasplaced into a microcentrifuge tube and centrifuged for 5 minutes at10,000 rpm. A 0.5 ml sample of the centrifuged liquid was acidified with0.5 ml of 10 mmol sulfuric acid. The acidified rumen fluid was filteredthrough a 0.2 um membrane filter (Gelman Laboratory Supor®-200).Volatile fatty acids were determined using a Bio Rad HPLC system. A 20ul sample was injected into an HPX 87H column using a 5 mmol sulfuricacid mobile phase. One ml/minute flow rate.

Analysis of the VFA from in vitro rumen fluid fermentation flasksidentified strains P169, P170, P179, P195 and P261 as the highestpropionate producing strains. All strains identified were from genotypegroup 1. The presence of the 7.0 or 2.7 kb plasmid did not effectpropionate production since strains that did not contain these plasmidsproduced similar levels of propionate. Strains P169, P170, P195, andP261 produced at least 0.9% (vol/vol) propionate in sodium lactate broth(NLB) and at least 0.2% (vol/vol) propionate in rumen fluid (in vitro).Strain P169 was used in subsequent animal trials described below.

Example 2 First Study of Isolates in Ruminants

Six non-lactating Holstein×Friesian dairy cows were fed a standard highforage diet (hay and grain, with a forage:grain ratio of 78/22). Cowshad constant access to forage, and the grain was offered in two feedsper day at 8 a.m. and 5 p.m.

Two successive periods of testing were used. In period 1, which wasweeks 1, 2, and 3, the cows were fed a control diet without P.acidipropionici strain P169. In period 2, which was weeks 4, 5, and 6,the cows were fed a control diet and the P. acidipropionici strain P169was introduced directly into the rumen under the fiber layer.

Throughout the six week experimental period, measurements were taken ofall food offered and all refusals. Cows were weighed at the start andend of each period. A pH measurement of the rumen fluid was taken.

At 08.00 h and 14.00 h, there was no effect of treatment(supplementation with Propionibacterium) on the rumen pH measured.However, in samples obtained at 19.30 h, cows that received thePropionibacterium had a significantly higher (P<0.01) rumen pH whencompared with cows that had not received the Propionibacterium. Theresults indicate that, whereas in the control group, there was a declinein rumen pH during the 11.5 h after the morning allocation ofconcentrate (−0.486 pH units), in the cows which received thePropionibacterium, the decline in rumen pH occurred during 6 h after themorning feed (−0.32 pH units) and then remained relatively stable beforereturning to the prefeeding value.

While applicants do not wish to be restricted to a particular theory ofthe cause of the pH changes observed in the 3 animals, the followingtheory is one possible explanation. Lactate, which is a byproduct ofruminal digestion, was used by the Propionibacteria. Thus, thePropionibacterium had a physiological effect on the cows.Propionibacteria use lactate to form propionate and acetate, which areweaker acids than lactate. It is expected that if the rumen were testedat a later time, then it would show an increase propionate level.

Example 3 Second Study of Isolates in Ruminants

Nineteen pluriparous cows were assigned randomly to one of two dietarygroups: total mixed ration (TMR) without Propionibacteria (control,n=10) or TMR plus Propionibacteria (Treated, n=9) from −2 wk to 12 wkpostpartum. The cows were allowed free access to feed and water. Eachtreated cow received 17 g of a 1:10 preparation of the freeze-driedpropionibacteria culture (strain P169 at 3.5×10¹⁰ CFU/g) andmaltodextrin carrier (strain P169) daily top-dressed into 1 to 2 kg ofthe TMR. Cows were individually fed and housed in a stanchion barn andgrouped by treatment to prevent potential transfer of Propionibacteriafrom treated to control cows. Half of each group of cows was placedacross each other and two unoccupied stalls with a plywood partitionseparated each adjacent group of cows. Each day cows were placed in adry lot for two 5 to 6 h intervals (800 to 1300 and 2100 to 3000). OnePropionibacteria-treated cow was taken out of the study due to a footproblem leaving nine cows in the treated group. Weekly BW were recordedand body condition of the cows were evaluated on wk 4 and 10 postpartumusing a five point scale 1=very thin to 5=excessively fat.

The TMR was composed of sorghum silage, alfalfa hay, sorghum\sudansilage, whole cottonseed, and concentrate. Energy concentration of thediet was formulated to support daily milk production of 50 kg (NRC,1989). Daily feed intake was recorded and the diet was sampled weeklyand composited monthly for analysis.

Cows were milked twice daily (0300 and 1500 h), and milk yield wasrecorded. Milk samples were collected weekly during successive a.m. andp.m. milkings and analyzed for percent milk fat, protein, lactose,solids-non-fat (SNF), somatic cell count (SCC) and milk urea nitrogen(MUN). Milk production was corrected for percent milk fat (fat correctedmilk (FCM)).

Blood samples (10 ml) were collected twice weekly via coccygealvenipuncture. After collection in tubes containing EDTA, blood wascentrifuged at 1200×g for 20 min (5° C.), and plasma was decanted andstored frozen at −20° C. for subsequent analysis.

Milk Protein: There was a significant interaction (P<0.001) betweentreatment and week postpartum on percent milk protein.Propionibacteria-treated cows had higher (P<0.001) percent milk proteinon wk 1 of lactation than control cows but not in the subsequent weeks(FIG. 3). Percent milk protein decreased from wk 1 to 3 and plateauedfrom wk 4 to 12 in both groups of cows (FIG. 3).

Solids-Non-Fat: There was a significant interaction between treatmentand week postpartum (P<0.05) on percent milk SNF.Propionibacteria-treated cows had higher (P<0.001) percent SNF on wk 1of lactation than control cows but not during the following weeks (FIG.4). Percent SNF decreased from wk 1 to 3 and remained stable in thesubsequent weeks in both groups of cows (FIG. 4).

Milk Fat: There was a significant interaction between treatment and weekpostpartum on percent milk fat The average milk fat percentage tended todiffer (P=0.13) between Propionibacteria-treated (3.2±0.08%) and control(3.02±0.08%) cows. (FIG. 5).

Dry Matter Intake: DMI expressed as g DMI per kg body weight (BW)differed significantly (P<0.01) between Propionibacteria-treated andcontrol cows. Averaged over the 12-wk period, control andPropionibacteria-treated cows consumed 23.97±0.48 and 23.37±0.50 kg/d,respectively. However, treated cows weighed more (667.1 kg±19 kg) thancontrol cows (616.2±18 kg) and when expressed on a body weight basis wassignificantly different (FIG. 6).

Fat Corrected Milk: The interaction of treatment and week postpartum didnot affect (P>0.50) FCM. FCM production did not differ between thecontrol and treated cows over the 12-wk study. Control andPropionibacteria-treated cows produced 34.49 t 0.86 and 35.16±0.89 kg/d,respectively. (FIG. 7) The FCM results show that cows did not decreasein production when they consumed less.

Glucose and Insulin: Plasma concentrations of insulin were determined byusing solid-phase insulin RIA kit (Micromedic Insulin Kit, ICNBiomedicals, Costa Mesa, Calif.) except that bovine insulin was used asa reference standard (25.7 IU/mg) as previously described (Simpson etal., 1994). Intraassay and interassay coefficients of variation were12.8% and 7.8%, respectively.

Plasma concentrations of glucose were determined using Glucose kits(Roche Diagnostic Systems, Inc., NJ) and a clinical analyzer (Cobas FARAII, Roche Analytical Instrument, Montclaire, N.J.). This procedure wasbased on the hexokinase coupled with glucose-6-phosphate dehydrogenaseenzymatic reaction. The intraassay coefficient of variation was 2.3%.

Neither plasma glucose (P>0.10) nor plasma insulin (P>0.50)concentrations were affected by the interaction of treatment×weekpostpartum. Cows fed Propionibacteria had an average plasma glucoseconcentration 60.00±0.91 mg/dl comparable (P>0.50) to that of controlcows 60.02±0.88 mg/dl. Concentrations of glucose in plasma increased(P<0.01) with wk postpartum such that wk 2 average glucose concentrationwas higher by 24% (P<0.02) than wk 1. Plasma glucose level did notchange significantly after wk 2 postpartum, indicating that reproductivefunction was maintained.

Average plasma concentration of insulin in cows fed Propionibacteria wassimilar (P>0.10) to the control cows (0.39±0.02 vs. 0.42±0.02 ng/ml).Concentrations of insulin in plasma increased (P<0.001) with weekpostpartum such that insulin concentrations at wk 2 differed from wk 1(P<0.10). Plasma insulin concentrations increased gradually thereaftersuch that over the 12-wk period, plasma insulin increased twofold(0.25±0.03 ng/ml in wk 1 to 0.51±0.03 ng/ml in wk 12), indicating thatreproductive function was maintained.

Non-Esterified Fatty Acids and Leptin: NEFA concentrations weredetermined by enzymatic method using NEFA-C kits (Waco Chemicals USA,Inc., VA) and a clinical analyzer (Cobas FARA II, Roche AnalyticalInstrument, Montclaire, N.J.). This enzymatic method utilizes acyl-CoAsynthethase and acyl-CoA oxidase to produce3-methyl-N-ethyl-N-(B-hydroxyethyl)aniline (MEHA). The intraassaycoefficient of variation was 4.5%.

Leptin plasma concentrations were measured using a Multi-species RIA kitassay (LINCO Research, Inc., St. Charles, Mo.) according to themanufacturer's recommendations with minor modifications. Briefly, on thefirst day, 100 Al of first antibody were added to all tubes except totalcount (TC) and non-specific binding (NSB) tubes then vortexed, coveredand incubated for 24 h at 4° C. The standard curve was modified toinclude 1, 2, 3, 5, 10 and 20 ng/ml of human leptin standard. On thesecond day, 100 pl of the tracer (1 251-human leptin) was added to alltubes then incubated for another 24 h at 4° C. On the third day, 1.0 mlof precipitating reagent was added to all tubes except TC tubes thenincubated for 20 min at 4° C. Tubes were centrifuged at 3,000×g for 30min, then supernatant was decanted and precipitate was counted using theGamma Counter. The sensitivity of the assay as defined as 95% of totalbinding was 0.85±0.08 ng/ml.

There was a significant (P<0.01) treatment×week postpartum interactionon plasma NEFA concentrations. Plasma NEFA concentrations ofPropionibacteria-treated cows at wk 1 postpartum were greater (P<0.01)than control cows (FIG. 9). Plasma NEFA concentrations decreased(P<0.001) with week postpartum for both groups of cows, although thedecrease was more dramatic in Propionibacteria-treated than control cows(FIG. 8), indicating that cows that were fed Propionibacteria weremoving towards positive energy balance.

Plasma leptin concentrations were significantly different (P<0.10) inPropionibacteria-treated cows (8.10±1.0 ng/ml) compared to control cows(5.25±1.0 ng/ml) (FIG. 9), demonstrating that the direct fedPropionibacteria had a positive effect on cows' plasma leptin, which maybe an important signal for regulation of feed consumption thatindirectly affects milk production, energy status, and reproductivefunctions. Treatment×week interaction (P>0.50) and week postpartum(P>0.50) did not affect plasma leptin concentrations (FIG. 9).

EB Calculations: EB was calculated by using net energy intake as theaverage daily DMI multiplied by the net energy concentration of thediet. Net energy required for daily maintenance of the animals wasderived using the equation 80×BW '75 (kg)/1000 (NRC, 1989). Daily energyfor milk production was calculated using the formula (Tyrell and Reid,1965), Milk yield (kg)×[92.239857 (% milk fat)+49.140211 (%SNF)−56.393297]/1000 where milk yield is average daily yield for theweek, and milk composition based on weekly milk analysis. This equationreflects the metabolic status of the cow more accurately than theconventional method of measuring milk yield alone (Butler and Smith,1989).

EB was not influenced (P>0.10) by interaction of treatment×weekpostpartum, but EB was affected (P<0.001) by week postpartum andtreatment (P<0.10) (FIG. 10). Generally, both groups of cows gained apositive EB starting 8 wk of lactation. Specifically, postpartum weeks1, 3 and 6 differed (P<0.05) from their succeeding week by −3.1, −2.5,and −2.27 Mcal/d, respectively (FIG. 10). Average EB of postpartum cowstended to differ (P<0.10) between cows fed with Propionibacteria(−1.596±0.72 Mcal/d) and control cows (0.196±0.69 Mcal/d) during the12-wk feeding period.

The interaction of treatment×week postpartum did not affect (P>0.50)body weight (BW). During the first 12 wk of lactation, average BW ofpostpartum cows tended to differ (P<0.10) betweenPropionibacteria-treated (667.1 kg±19 kg) and control (616.2±18 kg)cows. Also, weekly BW differed (P<0.001) among weeks postpartum. In bothgroups of cows, BW decreased between wk 1 and wk 3 but did notsignificantly change between wk 5 and wk 12 of lactation (data notshown).

The interaction (P>0.50) between treatment×week postpartum did notaffect average body condition score (BCS). Also, treatment had no affect(P>0.50) on BCS, measured at wk 4 and 10 postpartum. The BCS ranged fromwas 2.5 to 3.75 and averaged 2.69± for control cows and 2.68±0.06 forPropionibacteria-treated cows. Average weekly BCS increasedsignificantly (P<0.01) from wk 4 to wk 10 (2.53±0.07 vs. 2.86±0.06) inboth groups of cows.

It is understood that the various preferred embodiments are shown anddescribed above to illustrate different possible features of theinvention and the varying ways in which these features may be combined.Apart from combining the different features of the above embodiments invarying ways, other modifications are also considered to be within thescope of the invention. For example, the invention is applicable toother lactating ruminants, such as sheep and goats.

The invention is not intended to be limited to the preferred embodimentsdescribed above, but rather is intended to be limited only by the claimsset out below. Thus, the invention encompasses all alternate embodimentsthat fall literally or equivalently within the scope of these claims.

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1-17. (canceled)
 18. A method of feeding a first ruminant, the methodcomprising: feeding an effective amount to colonize the rumen of thefirst ruminant the Propionibacterium acidipropionici strain P169 ATCCPTA-5271 to the first ruminant; and after the feeding, testing the firstruminant for a least one of energy balance, plasma non-esterified fattyacids levels, glucose levels, propionate levels, and plasma leptinlevels, wherein the feeding of the strain increases at least one ofenergy balance, plasma non-esterified fatty acids levels, glucoselevels, propionate levels, and plasma leptin levels in the firstruminant when compared to the respective energy balance, plasmanon-esterified fatty acids levels, glucose levels, propionate levels,and plasma leptin levels in a second ruminant not fed the strain. 19.The method of claim 18, wherein the energy balance is increased.
 20. Themethod of claim 18, wherein the plasma non-esterified fatty acids levelsare increased.
 21. The method of claim 18, wherein the plasma leptinlevels are increased.
 22. The method of claim 18, wherein the glucoselevels are increased.
 23. The method of claim 18, wherein the propionatelevels are increased.
 24. The method of claim 18, wherein the first andsecond ruminants are bovines.
 25. The method of claim 18, wherein thefirst ruminant is fed the strain at a level such that the first ruminantis dosed daily with about 6×109 CFU to about 6×1012 CFU/animal/day. 26.The method of claim 25, wherein the first ruminant is fed the strain ata level such that the ruminant is dosed daily with about 6×1011CFU/animal/day.
 27. The method of claim 18, wherein the first ruminantis fed the strain until populations of 105 to 108 CFU/ml ruminal fluidare established in the rumen of the first ruminant.
 28. The method ofclaim 18, wherein the first ruminant is fed the strain from −2 to 12weeks postpartum.